comprehensive study of valuable lipophilic phytochemicals

Comprehensive Study of Valuable Lipophilic Phytochemicalsin Wheat BranPepijn Prinsen,† Ana Gutierrez,† Craig B. Faulds,‡,§ and Jose C. del Río†,*†Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, E- 41080 Seville, Spain‡Aix Marseille Universite, POLYTECH Marseille, UMR 1163 Biotechnologie des Champignons Filamenteux, 163 avenue de Luminy,13288 Marseille cedex 09, France§INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, 163 avenue de Luminy, 13288 Marseille cedex 09, France

ABSTRACT: Wheat bran, the major side-stream generated in the milling of wheat grains in the production of white flour,contains significant quantities of carbohydrate and proteins. While not interfering with flour utilization, the bran could beconsidered as an important feedstock within a biorefinery concept. Wheat bran also contains some amounts of lipids that can beused as a source of valuable phytochemicals. Gas chromatography and mass spectrometry analysis of the lipid composition ofdestarched wheat bran demonstrated that the predominant lipids found in wheat bran were free fatty acids (ca. 40% of totallipids), followed by acylglycerols (40%). Additionally, important amounts of alkylresorcinols (13% of total lipids) and steroidcompounds (hydrocarbons, ketones, free sterols, sterol glycosides, sterol esters, and sterol ferulates) (7% of total lipids) were alsopresent among the lipids of wheat bran. The use of wheat bran as a valuable source of phytochemicals of interest in the context ofa wheat bran biorefinery is discussed.

KEYWORDS: wheat bran, lipids, alkylresorcinols, steroids, triglycerides

■ INTRODUCTION

Wheat (Triticum spp.) is among the most extensively cultivatedcrop in the world, and is the most widely grown and consumedcereal by humans.1 Whole-grain wheat is composed of 10−14%bran, 2.5−3.0% germ, and 80−85% endosperm.2 Wheat branis the hard outer layer of a wheat grain and consists of thecombined aleurone and pericarp. Wheat bran is producedin enormous quantities as a major byproduct of the millingprocess for the production of white wheat flour, with an annualestimated production of 90 million tons.3

Nowadays, wheat bran is mainly used as a low valueingredient for both human and animal feed.4 However, due tothe enormous quantities produced in the milling industry andits specific properties, wheat bran has also been considered tohave an enormous potential as a valuable and versatile feed-stock for future biorefineries3 or as a value-added stream ofexisting milling facilities. The development of such biorefinerysystems utilizing agro-industrial side-streams or byproducts areessential for ensuring the sustainable supply of food, feed,chemicals, materials, goods, fuels, and energy in the future.Commercially produced wheat bran contains relatively lowamounts of cellulose (16%) and lignin (6.6%), but presentsimportant amounts of glucuronoarabinoxylans (38%), proteins(25%), and starch (9%).3,5 However, wheat bran also containssignificant amounts (up to 4%) of lipids that can also be usedas a source of valuable phytochemicals.3 It is known thatphytochemicals present in grains have the potential to reducesome diseases, including cardiovascular diseases,6 diabetes,7

and cancer.8,9 Some of the mentioned health benefits may beattributed to the antioxidant activity of phenolic compoundssuch as ferulic acid, other polyphenols (lignans, anthocyaninsand alkylresorcinols), carotenoids, and vitamin E.2 In particular,wheat bran has been widely recognized as a cancer preventive

agent, although the mechanism of the protection is not yet fullyunderstood.9 The lipophilic extractives of wheat bran are knownto be cytotoxic,9,10 and in particular alkylresorcinols have beenfound to exert higher cytotoxic effects on cancer cells.11

However, and despite its high significance, there are onlylimited studies reporting the complete and detailed chemicalcomposition of lipids in wheat bran. Alkylresorcinols have beenamong the most widely reported lipid compounds in wheatbran,9,11−14 although other lipid constituents, such as fattyacids and sterols, have also been reported.9,12 In this work, weperformed a comprehensive characterization of the lipophiliccompounds occurring in wheat bran by the use of gas chromato-graphy (GC) and gas chromatography−mass spectrometry(GC-MS), according to the method previously described.15

This study can provide significant further knowledge about thecomposition of lipids of wheat bran, which can be regarded as avaluable source of active phytochemicals.

■ MATERIALS AND METHODSSamples. The wheat bran used for this study consisted of a

commercial destarched sample obtained from Agro-industrieRecherches et Developments (Pomacle, France). The sample wasstored at room temperature in an opaque sealed container, with amoisture content of 8%, until further processing. Additional informationregarding the bulk composition of this sample has been previouslypublished.5 The wheat bran sample was subsequently air-dried andknife-milled (Janke and Kunkel, Analysenmuhle), and the lipophiliccompounds extracted with acetone for 8 h in a Soxhlet apparatus.The acetone extracts were evaporated to dryness, and resuspended in

Received: October 23, 2013Revised: January 20, 2014Accepted: January 22, 2014Published: January 22, 2014

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chloroform for chromatographic analysis of the lipid fraction. Theacetone-extracted sample was then extracted with hot water (100 mL,3 h at 100 °C) to determine the water-soluble material. Klason lignincontent was estimated as the residue after sulphuric acid hydrolysis ofthe pre-extracted material, corrected for ash and protein content,according to the TAPPI method T222 om-88.16 The acid-soluble ligninwas determined, after the insoluble lignin was filtered off (Duran filtercrucible 4; nominal pore size max. 10−16 μm), by UV-spectroscopicdetermination at 205 nm wavelength using 110 L cm−1 g−1 as theextinction coefficient. Holocellulose was isolated from the pre-extractedfibers by delignification for 4 h using the acid chlorite method.17 Theα-cellulose content was determined by removing the hemicellulosesfrom the holocellulose by alkali extraction.17 Ash content was estimatedas the residue after 6 h of heating at 575 °C according to the TAPPImethod T211 om-02.16 Three replicates were used for each sample.

GC and GC-MS Analyses. The lipophilic extractives (0.8 mg)were silylated with 0.250 mL bis(trimethylsilyl)trifluoroacetamide(BSTFA, from Supelco) in the presence of 0.050 mL pyridine at70 °C for 2 h before GC and GC-MS analyses. An HP 5890 gaschromatograph (Hewlett-Packard, Hoofddorp, Netherlands) equippedwith a split−splitless injector and a flame ionization detector (FID)was used for GC analyses. The injector and the detector temperatureswere set at 300 and 350 °C, respectively. Samples were injected in thesplitless mode at a concentration of 7.5 mg/mL. Helium was used asthe carrier gas. The capillary column used was a high temperature,polyimide coated fused silica tubing DB5-HT (5 m x 0.25 mm I.D.,0.1 μm film thickness; J&W Scientific). The oven was temperature-programmed from 100 °C (1 min) to 350 °C (3 min) at 15 °C min−1.Peaks were quantified by area, and a mixture of different standards, suchas octadecane (Sigma-Aldrich, 99%), palmitic acid (Sigma-Aldrich,99%), sitosterol (Calbiochem, 98%), cholesteryl stearate (Sigma-Aldrich,99%), sitosteryl 3β-D-glucopyranoside (Matreya LLC, a mixture of threesteryl glucosides of 98% purity, of which 56% correspond to sitosterylglucoside) and triheptadecanoin (Sigma-Aldrich, 99%), with aconcentration range between 0.1 and 1 mg/mL, was used to elaboratecalibration curves. The correlation coefficient was higher than 0.99 in allcases. Quantification was obtained using response factors of the same orsimilar compounds (in the case of alkylresorcinols, they were quantifiedagainst sitosterol). The data from the three replicates were averaged.In all cases, the standard deviations from replicates were below 10%of the mean values. The total amounts of the different lipid familieswere determined by adding up the amounts of their constituentcompounds.

The GC-MS analyses were performed on a Varian Star 3400 gaschromatograph (Varian, Walnut Creek, CA) coupled with an ion-trapdetector (Varian Saturn 400) equipped with a high-temperaturecapillary column (DB-5HT, 15 m × 0.25 mm i.d., 0.1 μm film

Table 1. Abundance of the Main Constituents(% Dry-Weight) of Wheat Bran

constituent contenta

water-soluble extractives 2.4 ± 0.1lipids 2.0 ± 0.1Klason ligninb 12.1 ± 0.8acid-soluble lignin 8.5 ± 1.0carbohydrates 64.0 ± 1.0(α-cellulose) (30 ± 1.0)proteinsc 9.9 ± 1.0ash 1.1 ± 0.1

aAverage of three replicates. bCorrected for proteins and ash.cDetermined indirectly by subtracting the other components to 100%.

Figure 1. GC-MS chromatogram of the lipid extracts from wheat bran (as TMS-ether derivatives). F(n): n-fatty acid series; MG(n): monoglycerides;AR(n): n-alkylresorcinols; n denotes the total carbon atom number. Other compounds reflected are as follows: 1: campesterol; 2: campestanol; 3:stigmasterol; 4: sitosterol; 5: campesteryl 3β-D-glucopyranoside; 6: stigmasteryl 3β-D-glucopyranoside; 7: sitosteryl 3β-D-glucopyranoside; 8:campestanyl ferulate; and 9: stigmastanyl ferulate.

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Table 2. Composition and Abundance (mg/kg Fiber, d.a.f.) of Main Lipids Identified in the Extracts of Wheat Bran

compound abundance

n-fatty acids 6620n-tetradecanoic acid 40n-pentadecanoic acid 32n-hexadecanoic acid 1595n-heptadecanoic acid 42cis,cis-octadeca-9,12-dienoic acid 1500cis-octadec-9-enoic acid 1590n-octadecanoic acid 720n-nonadecanoic acid 33n-eicosanoic acid 190n-heneicosanoic acid 23n-docosanoic acid 285n-tricosanoic acid 66n-tetracosanoic acid 180n-pentacosanoic acid 35n-hexacosanoic acid 130n-heptacosanoic acid 8n-octacosanoic acid 98n-nonacosanoic acid 3n-triacontanoic acid 23n-hentriacontanoic acid 2n-docotriacontanoic acid 20n-tritriacontanoic acid 2n-tetratriacontanoic acid 3monoglycerides 25802,3-dihydroxypropyl tetradecanoate 202,3-dihydroxypropyl pentadecanoate 62,3-dihydroxypropyl hexadecanoate 9302,3-dihydroxypropyl heptadecanoate 72,3-dihydroxypropyl octadec-9,12-dienoate 5002,3-dihydroxypropyl octadec-9-enoate 7852,3-dihydroxypropyl octadecanoate 2002,3-dihydroxypropyl nonadecanoate 32,3-dihydroxypropyl eicosanoate 142,3-dihydroxypropyl heneicosanoate 22,3-dihydroxypropyl docosanoate 202,3-dihydroxypropyl tricosanoate 62,3-dihydroxypropyl tetracosanoate 252,3-dihydroxypropyl pentacosanoate 82,3-dihydroxypropyl hexacosanoate 322,3-dihydroxypropyl heptacosanoate 32,3-dihydroxypropyl octacosanoate 152,3-dihydroxypropyl nonacosanoate 12,3-dihydroxypropyl triacontanoate 3diglycerides 21501,2-dipalmitin 501,3-dipalmitin 1721,2-palmitoyl linolein 1401,2-palmitoyl olein 2101,2-palmitoyl stearin 501,3-palmitoyl linolein 1541,3-palmitoyl olein 2801,3-palmitoyl stearin 901,2-dilinolein 701,2-linoleoyl olein 951,2-linoleoyl stearin + 1,2-diolein 1201,2-oleoylstearin 501,2-distearin 501,3-dilinolein 951,3-linoleoyl olein 1201,3-linoleoyl stearin + 1,3-diolein 2501,3-oleoyl stearin 641,3-distearin 90

compound abundance

triglycerides 1960tripalmitin 10dipalmitoyl linolein 150dipalmitoyl olein 110dilinoleoyl palmitin 480linoleoyloleoyl palmitin 300dioleoyl palmitin 20trilinolein 250dilinoleoyl olein 400dilinoleoyl stearin 200tristearin 40n-alkanes 40n-tricosane 2n-pentacosane 5n-hexacosane 1n-heptacosane 10n-octacosane 1n-nonacosane 14n-triacontane 1n-hentriacontane 5n-tritriacontane 1sterols 510campesterol 120campestanol 45stigmasterol 35sitosterol 290stigmastanol 20sterol glycosides 135campesteryl-β-D-glucopyranoside 42stigmasteryl-β-D-glucopyranoside 8sitosteryl-β-D-glucopyranoside 85sterol esters 240campesteryl hexadecanoate 20campestanyl hexadecanoate 13stigmasteryl hexadecanoate 3sitosteryl hexadecanoate 60stigmastanyl hexadecanoate 20campesteryl octadec-9,12-dienoate 33campestanyl octadec-9,12-dienoate 7stigmasteryl octadec-9,12-dienoate 3sitosteryl octadec-9,12-dienoate 70stigmastanyl octadec-9,12-dienoate 11sterol ferulates 105campestanyl ferulate 67stigmastanyl ferulate 38steroid hydrocarbons 60ergostatriene 5ergostadiene 10stigmasta-4,22-diene 5stigmasta-3,5,22-triene 20stigmasta-3,5-diene 20steroid ketones 80stigmasta-4,22-dien-3-one 15stigmasta-3,5-dien-7-one 45stigmast-4-en-3-one 20n-alkylresorcinols 22505-n-pentadecylresorcinol 55-n-heptadecylresorcinol 805-n-nonadecylresorcinol 6605-n-heneicosylresorcinol 11405-n-tricosylresorcinol 2705-n-pentacosylresorcinol 905-n-heptacosylresorcinol 5



thickness; J&W Scientific). Helium was used as carrier gas at a rateof 2 mL/min. The samples, at concentrations of 2.5 mg/mL, wereinjected with an autoinjector (Varian 8200) directly onto the columnusing a SPI (septum-equipped programmable injector) system. Thetemperature of the injector during the injection was 60 °C, and0.1 min after injection was programmed to 380 °C at a rate of 200 °Cmin−1 and held for 10 min. The oven was heated from 120 °C(1 min) to 380 °C (5 min) at 10 °C min−1. The temperature of thetransfer line was set at 300 °C. Compounds were identified bycomparing their retention times and mass spectra with authenticstandards, except for alkylresorcinols and sterol ferulates, which weretentatively identified by comparing their mass spectra with thosereported in the literature. Single ion chromatographic profiles wereused to estimate compound abundances when two peaks partiallyoverlapped.

■ RESULTS AND DISCUSSION

Lipid Composition of Wheat Bran. The destarched wheatbran sample selected for this study is mostly composed ofcarbohydrates (64.0% of the whole material, among which30% corresponds to α-cellulose), lignin (20.6%), and proteins(9.9%), as seen in Table 1. Minor amounts of water-solubleextractives (2.4%), lipids (2.0%), and ash (1.1%) were alsopresent in this sample. The detailed analysis of the chemicalcomposition of the lipids was accomplished by GC and GC-MS(after preparation of the trimethylsilyl (TMS)-ether derivatives)according to the method previously described.15 The chromato-graphic conditions used for the analyses, by using short- andmedium-length high-temperature capillary columns with thin

Figure 2. Structures representative of the main aliphatic lipophilic compounds identified in the extracts of wheat bran and referred in the text. (I)hexadecanoic (palmitic) acid; (II) 9-octadecenoic (oleic) acid; (III) 9,12-octadecadienoic (linoleic) acid; (IV) 2,3-dihydroxypropyl hexadecanoate(1-monopalmitin); (V) 2,3-dihydroxypropyl octadecenoate (1-monoolein); (VI) 2,3-dihydroxypropyl octadecadienoate (1-monolinolein); (VII)1,2-palmitoyl olein; (VIII) 1,3-palmitoyl olein; (IX) dilineoylpalmitin; (X) n-nonacosane; and (XI) 5-n-heneicosylresorcinol.



films, allow for the elution of a wide range of compounds, fromlow molecular weight free fatty acids and sterols to intact highmolecular weight lipids such as sterol esters, sterol glycosides,or triglycerides, that are usually biased in more standard GCconditions.Figure 1 shows the GC-MS chromatogram of the lipids (as

their TMS-ether derivatives) extracted from wheat bran. A largenumber of compounds were identified and quantified. Theidentities and abundances (mg/kg fiber, dry, ash-free basis) ofthe main lipid compounds identified in the extracts of wheatbran are listed in Table 2. Structures representative of the mainlipid families identified in wheat bran, including aliphatic andsteroid compounds, are shown in Figure 2 and Figure 3. Theresults demonstrated that the predominant lipids identifiedamong the extracts of this wheat bran sample were series of freefatty acids, accounting for around 40% of all identified compounds,followed by series of acylglycerols (40% in total), that included

mono-, di- and triglycerides. Important amounts of alkylresorci-nols (13% of all identified compounds) were also found amongthe lipids of wheat bran. Alkylresorcinols have been the mostwidely reported lipids in wheat bran.9,11−13 Steroid compounds(including hydrocarbons, ketones, free sterols, sterol glycosides,sterol esters, and sterol ferulates) were also present among thelipids of wheat bran in important amounts (7% of all identifiedlipids). Only free sterols and sterol ferulates were previouslyreported among the lipids of wheat bran.9,12,18 Lower amountsof other aliphatic series such as n-alkanes, were also observed.The distributions of the main aliphatic series (fatty acids,n-alkanes, monoglycerides, and n-alkylresorcinols) are repre-sented in the histograms of Figure 4.

Aliphatic Series. Free fatty acids (6620 mg/kg) were themost abundant family of identified lipids. The series of free fattyacids was found in the range from tetradecanoic acid (C14) totetratriacontanoic acid (C34), with a predominance of the even

Figure 3. Structures of the main steroid compounds identified in wheat bran and referred in the text. (XII) sitosterol; (XIII) campesterol; (XIV)stigmasterol; (XV) campestanol; (XVI) stigmastanol; (XVII) sitosteryl 3β-D-glucopyranoside; (XVIII) sitosteryl linoleate; (XIX) campestanylferulate; (XX) stigmastanyl ferulate; (XXI) stigmasta-3,5-dien-7-one; (XXII) stigmasta-4,22-dien-3-one; (XXIII) stigmasta-4-en-3-one; (XXIV)stigmasta-3,5,22-triene; and (XXV) stigmasta-3,5-diene.



carbon atom number homologues. Hexadecanoic acid (palmiticacid, I) was the most abundant free fatty acid (1595 mg/kg),together with the unsaturated cis-9-octadecenoic acid (oleicacid, II) (1590 mg/kg) and cis,cis-9,12-octadecadienoic acid(linoleic acid, III) (1500 mg/kg). High amounts of thesaturated octadecanoic acid (stearic acid) (720 mg/kg) werealso found, while the rest of fatty acids were present in minoramounts. Despite the high abundance of fatty acids identifiedin wheat bran, a detailed analysis of their occurrence has notbeen performed so far, and there is a substantial gap in theinformation reported in the literature. The major free fattyacids previously reported in wheat bran were linolenic (C18:3)and linoleic (C18:2) acids,9 which substantially differ from ourresults.Acylglycerols (including mono-, di-, and triglycerides) were

also found among the lipids of wheat bran in importantamounts, accounting for a total of 6490 mg/kg, with mono-glycerides being the predominant ones. However, and despitethis abundance, the detailed analyses of their componentshave not been performed so far, and only the occurrence ofthe diglyceride dilinoleoylglycerol was previously reported.9

Monoglycerides in wheat bran accounted for 2580 mg/kg, andwere identified in the range from 2,3-dihydroxypropyl tetra-decanoate (C14) to 2,3-dihydroxypropyl triacontanoate (C30),with a strong predominance of the even carbon atom numberhomologues. 1-Monopalmitin (IV) was the most abundantmonoglyceride (930 mg/kg), followed by the unsaturated1-monoolein (V, 785 mg/kg) and 1-monolinolein (VI,500 mg/kg), and the saturated 1-monostearin (200 mg/kg).To our knowledge, this is the first time that the series of

monoglycerides has been reported in wheat bran. Thedistribution of esterified fatty acids occurring as monoglyceridesroughly matches that of the free fatty acids, as seen in thehistograms of Figure 4. Diglycerides were also found in importantamounts (2150 mg/kg) among the lipids in wheat bran. Themost abundant diglycerides identified were 1,2-palmitoyl olein(VII) (210 mg/kg) and 1,3-palmitoyl olein (VIII) (280 mg/kg),with a predominance of the 1,3-isomers, as also occurs in othergrasses.19,20 Palmitic and linoleic acids were the most importantesterified fatty acids occurring as diglycerides, as also occurred inthe case of monoglycerides. A previous work only identifieddilineoylglycerol (although it did not report the substitutionpattern), but these compounds are minor components in ourstudy. Triglycerides were also identified in high amounts amongthe lipids of wheat bran, accounting for 1960 mg/kg, (Table 2)and, although their occurrence was previously reported,9 this isthe first time that the identification of individual triglycerideswas achieved by GC-MS. Dilinoleoyl palmitin (IX) was the mostabundant triglyceride in the wheat bran sample (480 mg/kg)followed by dilinoleoyl olein (400 mg/kg), linoleyloleyl palmitin(300 mg/kg), and trilinolein (250 mg/kg). Palmitic and linoleicacids were the most abundant esterified fatty acids presentas triglycerides, as also occurred in the case of mono- anddiglycerides.A series of n-alkanes could also be identified among the lipids

of wheat bran, although in lower amounts (40 mg/kg). Theseries of n-alkanes was found in the range from n-tricosane (C23)to n-tritriacontane (C31), with a strong odd-over-even atomcarbon number predominance. Nonacosane (X) was the maincompound of this series, accounting for 14 mg/kg, followed by

Figure 4. Distribution of the main aliphatic series identified in the extracts of wheat bran. (a) n-Fatty acids; (b) n-alkanes; (c) monoglycerides; and(d) n-alkylresorcinols. The histograms are scaled up to the abundance of the major compound in the series.



heptacosane (10 mg/kg). The rest of the alkanes were identifiedin very minor amounts.Steroid Compounds. Different families of steroid compounds

were identified among the lipids in wheat bran, including steroidhydrocarbons, steroid ketones, free sterols, sterol glycosides,sterol esters, and sterol ferulates. Free sterols, accounting for510 mg/kg, were the major family of steroid compounds presentin wheat bran, with sitosterol (XII) occurring at the highest level(290 mg/kg), followed by campesterol (XIII) (120 mg/kg). Inaddition, lower amounts of stigmasterol (XIV) and the saturatedcampestanol (XV) and stigmastanol (XVI) were also identified.Sterols conjugated with carbohydrates forming sterol glycosidesor with long chain fatty acids or ferulic acid forming sterol esterswere also identified. Important amounts of sterol glycosides(135 mg/kg) were identified among the lipids of wheat bran bycomparison with the mass spectra and relative retention times ofauthentic standards.21 As far as we know, this is the first time thatsterol glycosides are reported as constituents of wheat bran lipids.Sitosteryl 3β-D-glucopyranoside (XVII) was the most predom-inant sterol glycoside in wheat bran (85 mg/kg) with loweramounts of campesteryl and stigmasteryl 3β-D-glucopyranosides,as usually observed in other grasses.19,20 Sterol esters of longchain fatty acids corresponding to campesterol, campestanol,stigmasterol, sitosterol, and stigmastanol esterified with differentfatty acids were also found (240 mg/kg). Each sterol ester seriesidentified showed two major peaks due to the esterification withthe C16 (palmitic acid) and with the unsaturated linoleic acid.

Sitosteryl linoleate (XVIII) was the most predominant(70 mg/kg) sterol ester in wheat bran, followed by sitosterylhexadecanoate (60 mg/kg), as also occurs in other cereals.22

Figure 5. Mass spectra of (a) campestanyl ferulate, and (b) stigmastanyl ferulate, (as TMS ethers).

Figure 6. Single ion chromatogram (m/z 266) showing the distributionof sterol ferulates (as TMS ethers) identified in the extracts of wheatbran. (a) Campestenyl ferulate; and (b) stigmastanyl ferulate.



Sterol ferulates were also identified among the lipids of wheatbran (105 mg/kg), and consisted exclusively of the saturatedcampestanyl ferulate (XIX) and sitostanyl ferulate (XX). Theidentification of sterol ferulates was obtained from the massspectra of their TMS ethers that show a characteristic base peakion at m/z 266, typical of these molecules, together with themolecular ions at m/z 650 and m/z 664 for campestanyl andstigmastanyl ferulates, respectively.23 The mass spectra of thesesterol ferulates are shown in Figure 5, whereas Figure 6 displaysthe single ion chromatogram (m/z 266) showing the distributionof the series of sterol ferulates present among the lipids of wheatbran. The identification of these compounds is consistent withprevious observations in several cereal bran oils.12 Differentsteroid ketones, namely stigmast-3,5-dien-7-one (XXI), stigmasta-4,22-dien-3-one (XXII) and stigmast-4-en-3-one (XXIII), werealso identified in lower amounts (80 mg/kg) among the lipids ofwheat bran. Steroid hydrocarbons were also found, albeit in low

amounts (60 mg/kg), and included stigmasta-3,5,22-triene(XXIV) and stigmasta-3,5-diene (XXV). The important amountsof steroid compounds present in wheat bran, and particularly thehigh content of free and conjugated sterols, makes wheat bran agood source of valuable phytosterols.

Alkylresorcinols. A series of 5-n-alkylresorcinos wasidentified among the lipids of wheat bran in important amounts(2250 mg/kg). The identification of 5-n-alkylresorcinols wasachieved from their mass spectra that show a base peak dueto the McLafferty rearrangement at m/z 268, characteristic ofthese molecules.14,24 The mass spectra of three representative5-n-alkylresorcinols are shown in Figure 7, with the character-istic base peak at m/z 268 and the molecular ions at m/z520, m/z 548, and m/z 576 for the TMS ether derivativesof 5-n-nonadecyl-, 5-n-heneicosyl-, and 5-n-tricosylresorcinols,respectively. Figure 8 displays the single ion chromatogram(m/z 268) showing the distribution of the series of

Figure 7. Mass spectra of selected alkylresorcinols (as TMS ethers): (a) 5-n-nonadecylresorcinol, (b) 5-n-heneicosylresorcinol, and (c)5-n-tricosylresorcinol.



5-n-alkylresorcinols present among the lipids of wheat bran.The 5-n-alkylresorcinols ranged from 5-n-pentadecyl (C15) to5-n-heptacosylresorcinol (C27), with the sole occurrence ofthe odd carbon atom-numbered homologues, and with5-n-heneicosylresorcinol (C21, XI) being the most abundantone (1140 mg/kg), followed by 5-n-nonadecylresorcinol(660 mg/kg). Alkylresorcinols are known to occur in cerealgrains and their occurrence in wheat bran has been widelyreported.9,11−14,25 The distribution of alkylresorcinols identifiedis similar to that found in previous works,9,11−14,25 althoughin the present work we have extended the series of identifiedalkylresorcinols up to C27.Possible Uses and Benefits of Wheat Bran Lipids. The

study shown here indicates that wheat bran is a promisingsource of phytochemicals, such as fatty acids, triglycerides,phytosterols, and alkylresorcinols. These compounds have awide range of nutraceutical, pharmaceutical, and cosmeticproperties and are also of interest for other industrial sectors.The particularly high content of free fatty acids (40%) andacylglycerols (40%) in wheat bran extracts makes this material aninteresting feedstock for biodiesel production, which is nowadaysessentially produced from oil seed crops.26 The potential use ofcereal brans, such as rice bran, as useful feedstocks for biodieselproduction, has recently being reported,27 although there couldbe some considerations if the material used in animal diets arediverted to fuel production. Among the fatty acids, wheat branalso contains important amounts of linoleic acid, an essentialunsaturated omega-6 fatty acid that must be consumed forproper health and is of interest for the food industry. Moreover,linoleic acid is also used in pharmaceutical and cosmetic productsand is considered to influence the metabolic processes in the skinand to promote the activity of vitamins A and E and recoverybarrier properties of stratum corneum.28 On the other hand,phytosterols, which are also present in important amounts inwheat bran, when used as functional ingredients in foods, reduceblood cholesterol levels by interfering with intestinal cholesterolabsorption.29,30 The efficacy of phytosterols as cholesterol-lowering agents have been shown when incorporated into fatspreads as well as other food matrices. In addition, phytosterols

have been combined with other beneficial dietary componentsto enhance their effect on risk factors of cardiovascular disease.Phytosterols appear not only to play an important role in theregulation of cardiovascular disease, but also to exhibit anticancerproperties.30 Hence, phytosterols, especially sitosterol, aresuggested to have a protective effect against the most commoncancers in the developed countries including colon, prostate, andbreast.12 In addition, sterol ferulates present anti-inflammatoryeffects and are also effective antitumor-promoters.31 Finally,the important amounts of alkylresorcinols present in wheatbran are of special value since they are involved in multiplebiological activities, including antimicrobial,32 antiparasitic,33,34

antioxidant,13,14,35 and antimutagenic activities.9,11,12,36

In conclusion, we report a comprehensive description of thechemical composition of lipids in wheat bran that opens up newpossibilities for a more complete industrial utilization of thisside-stream in the context of a wheat bran biorefinery. Due tothe large amounts produced annually worldwide, wheat brancan be viewed as an unexploited and promising source of highlyvaluable phytochemicals with potential health benefits andbiological activities. Variations in the chemical composition ofthe lipids, however, can be expected to occur among differentwheat bran samples from differences in wheat cultivars andmilling procedures; the screening and analysis of additionalwheat bran samples are therefore needed in order to evaluatethe divergences in the composition and content of lipids amongsamples from different sources.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +34-95-4624711; Fax: +34-95-4624002; E-mail: [email protected] study has been funded by the Spanish project AGL2011−25379 and the EU-project LIGNODECO (KBBE-244362).Pepijn Prinsen thanks the Spanish Ministry of Science andInnovation for a FPI fellowship.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Jorge Rencoret for technical assistance in the GC-MSanalyses.

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Figure 8. Single ion chromatogram (m/z 268) showing thedistribution of the series of n-alkylresorcinols (as TMS ethers)identified in the extracts of wheat bran. Cn indicates the carbon lengthof the alkyl chain.



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comprehensive study of valuable lipophilic phytochemicals

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